Microbial infections, such as bacterial infections, that cause disease in people and in animals such as livestock and companion animals are of clear and obvious concern throughout the world. For example, bovine mastitis is the most important infectious disease affecting both the quality and quantity of milk produced in the world. This disease affects virtually every dairy farm and has been estimated to affect 38% of all cows. The disease can cause destruction of milk-synthesizing tissues which reduces milk production and alters milk composition. In severe cases, the productive performance of dairy cattle may be diminished permanently.
Thus, mastitis continues to be the single greatest impediment to profitable dairy production. Losses associated with mastitis cost American dairy producers about 2 billion dollars per year and cost dairy producers worldwide an estimated 25 billion dollars per year.
In spite of the severe economic impact of this disease on the dairy industry, the only widely accepted methods of mastitis control are based on post-milking teat disinfection and antibiotic therapy. These methods have the disadvantages that they are often ineffectual and result in milk loss during such treatments. Additionally, there is a growing concern in the United States and worldwide about the presence of drug residues in the food supply and the potential consequences for public safety.
There is a pressing need for an effective, safe, and economical mastitis treatment and prophylactic therapy which will reduce the dairy industry's dependence on chemical agents to attempt to prevent or treat mastitis.
The case of mastitis is only an example. In addition to mastitis, there are countless other microbial diseases, including bacterial diseases, of cattle and other animals, for which a therapy to reduce the incidence and/or to treat existing cases is needed.
It has been established through research that interleukin-2 (IL-2) stimulated porcine and human NK (natural killer) cells are capable of non-specific killing of bacteria which is mediated by the secretion of porcine NK-lysin and human granulysin, respectively. A parasitic protozoan, Entamoeba histolytica, also is capable of killing bacteria through the generation of a pore-forming protein called amoebophore.
Despite the considerable evolutionary distance between single-celled parasitic organisms and mammalian lymphocytes, the bacterial effector proteins porcine NK-lysin, human granulysin, and amoebophore are related members of the same saposin-like family of proteins, referred to as “SAPLIP”s. The SAPLIPs are small glycoproteins, often derived from larger precursor proteins in vivo, that carry out diverse functions through association with lipid membranes. The amino acid sequences of the SAPLIP family members include highly conserved cysteine residues that form disulfide bonds and provide a stable structure to the SAPLIPs. The SAPLIP members also share the characteristic that their secondary protein structure is made up mostly of α-helices joined by loops.
It has been demonstrated in in vitro studies that IL-2 stimulated bovine NK-cells possess antibacterial activity that is not MHC restricted. See, Sordillo, L M, et al., J. Dairy Sci., 74:3370 (1991), incorporated in its entirety herein by reference. This activity has been reported to be carried out by a small, secreted protein that is effective in small quantities and is not toxic to host cells. See, Sordillo-Gandy et al., International Patent Application Publication No. WO 98/08534 (1998), incorporated in its entirety herein by reference. This protein was reported to be approximately 16 kD in size as determined by SDS-PAGE and was shown to have antibacterial activity against a wide range of bacteria, including both gram-negative and gram-positive bacteria, without causing harm to host cells.
Although Sordillo-Gandy discloses the isolation of this protein, because it is produced in very small amounts by stimulated NK cells, the isolation of the protein in the native form is both tedious and time consuming. These difficulties have presented an impediment to further study of the protein and to utilizing the isolated native protein for its antimicrobial activity. Thus, a need exists for a means of obtaining this protein by a means other than by isolation.